Recent studies in our laboratory have shown the occurrence of multiple forms of both constitutive and inducible cytochrome P-450 in rat hepatic mitochondria, some of which are expressed, or induced in a sex dominant fashion. Based on this, the objective of this renewal application is to continue the further characterization of these P-450 forms with respect to catalytic function, primary structure, gene organization, chromosome location and transcriptional and post transcriptional regulation. The following lines of experiments will be carried out to accomplish this goal: 1) The functional properties of the P-450 enzymes will be studied by in vitro reconstitution of enzyme activity using various physiological substrates like testosterone, progesterone, cholesterol, vitamin D3 and lauric acid, and also various xenobiotic compounds. The functional properties will be further ascertained by expressing the cDNA clones in monkey COS cells or in appropriate cell systems. 2) Search for additional P-450 using other P-450 inducers, their purification and characterization, and the development of monoclonal and polyclonal antibodies will be continued. 3) The ongoing efforts on the isolation of cDNA clones from the lambda expression libraries using a combination of synthetic oligonucleotide and the antibody probes, and DNA sequencing will be continued to gain insight on the primary structure and evolution of these P-450 forms. 4) The extent of induction, as well as the age and sex dependent patterns of induction of P-450 forms by xenobiotic inducers and steroid hormones will be studied in vivo under various physiological and hormonal states, and in vitro in primary hepatocytes, or in the established cell lines, wherever possible. 5) The genes for the cholesterol 26 hydroxylase, the female specific D3 25 hydroxylase and some of the xenobiotic inducible forms will be isolated and sequenced to understand the structural organization and mode of regulation.